PROJECT SUMMARY/ABSTRACT Unraveling The Mechanism Of Cryptic Sexual Cycle Development In Amoebae Using Comparative Genomics And Cytology The proposed research aims to unravel the cytological and genetic evidence underlying the cryptic sexual life cycle of two amoeboids: Cochliopodium and Acanthamoeba. Sexual reproduction is highly beneficial in eukaryotes by enabling them to increase genetic diversity, fitness and virulence as in pathogenic eukaryotes. However, sexuality in most microbial eukaryotes is poorly understood due to their diverse and complex life cycle. Unlike most other amoebae that are assumed to engage in sex during the dormant, cyst stage (a challenge for experimental study); the target taxon, Cochliopodium, undergoes nuclear fusion in the actively growing vegetative stage making it an ideal candidate for investigative work involving live experimentation. Our findings from previous work with Cochliopodium have enabled us to make a comparative study with the unknown aspects of the life cycle of a close relative opportunistic human pathogen, Acanthamoeba. This amoeba causes severe infection of the eye, brain and skin infections. Acanthamoeba is considered to reproduce asexually but recent genomic data indicated that it may engage in cryptic sex. However, there is no corroborative evidence from behavioral or cytological data to support this claim. In this proposal, we use the knowledge gained from Cochliopodium to study the life cycle of Acanthamoeba with a similar approach. The proposed work will test three hypotheses related to sexual development in Cochliopodium and Acanthamoeba. These hypotheses are based from our previous published, on-going research and new discoveries. We will use a combination of methods including comparative genomics, transcriptomics, genetic manipulations and optimized cytological techniques to tackle the proposed hypotheses. Addressing the proposed hypotheses will enable the following new discoveries: (a) uncover cytological signatures of the stages of sexual development and (b) shed light on the molecular mechanisms of the cryptic sexual life cycle in Cochliopodium and Acanthamoeba; (c) patterns of inheritance in Cochliopodium; (d) setting a groundwork for understanding the evolution of sexuality in microbial eukaryotes with cryptic life cycles; (e). exploration of targets that might be used to combat eukaryotic pathogens.